Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3

ABSTRACT

This invention encompasses novel chimeric glycoproteins which are useful for preparing virus specific immune responses against human parainfluenza virus type 3, PIV3. Host cells transformed with structural genes coding for the glycoproteins, expression and replication plasmids containing the structural genes, vaccines made from the glycoproteins and methods for protecting humans by inoculation with said vaccines are also part of this invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. Ser. No. 07/582,891, filed Oct.5, 1990, now U.S. Pat. No. 5,169,628, which was the U.S. National Phaseapplication of PCT/US89/00814, filed Mar. 3, 1989, which was acontinuation of U.S. Ser. No. 07/184,648, filed Apr. 22, 1988,abandoned.

FIELD OF THE INVENTION

This invention encompasses novel chimeric glycoproteins which are usefulfor preparing virus specific immune responses against humanparainfluenza virus type 3, PIV3. Host cells transformed with structuralgenes coding for the glycoproteins, expression and replication plasmidscontaining the structural genes, vaccines made from the glycoproteinsand methods for protecting humans by inoculation with said vaccines arealso part of this invention.

BACKGROUND

Human parainfluenza virus type 3, PIV3, is an important primary cause ofsevere lower respiratory tract disease in infants and young children.The virus occurs worldwide and infects virtually all children under theage of four. Acute respiratory disease and secondary complications areparticularly serious in infants and young children due to the immaturityof the respiratory system and may require hospitalization in severecases. Lower respiratory infections are referable to all segments of therespiratory tract, are usually associated with fever, cough, runny nose,and fatigue, and are diagnosed clinically as bronchitis, bronchiolitis,pneumonia, croup, or viral infection. Older children and adults are alsofrequently reinfected although reinfection typically results in lesssevere upper respiratory tract illness.

Attempts to develop effective PIV3 vaccines have been largelyunsuccessful. Clinical studies using live or inactivated PIV3 vaccinesdemonstrated an increase in virus specific serum antibodies but providedno significant protection against the disease.

INFORMATION DISCLOSURE STATEMENT

The recombinant vaccinia virus expression system is known to separatelyexpress the F and HN glycoproteins of PIV3 and to separately induceprotective immune responses in challenged cotton rats, Collins, P. L.,et al, Expression of the F and HN Glycoproteins of Human ParainfluenzaVirus Type 3 by Recombinant Vaccinia Viruses: Contributions of theIndividual Proteins to Host Immunity, Journal of Virology 61: 3416-3423(1987). The recombinant vaccinia virus expression system is also knownto induce PIV3-specific serum neutralizing antibodies and to conferresistance to PIV3 replication in the respiratory tract in primates,Collins, P. L., et al., Journal of Virology 62: 1293-1296 (1988).Immunization with a mixture of purified F and HN glycoproteins inducedvirus neutralizing activity and afforded complete protection fromchallenge infection in hamsters, Ray, R., et al., Journal of Virology62: 783-787 (1988).

SUMMARY OF THE INVENTION

This invention encompasses a polypeptide comprising a signal sequenceand at least one immunogenic fragment from both human parainfluenzavirus type 3 glycoproteins F and HN. The use of this protein as avaccine, methods to prevent PIV3-related disease and preparation of thisprotein using recombinant techniques are also part of this invention.

DETAILED DESCRIPTION

The following defined terms are used in this specification. The phrase"cell culture" refers to the containment of growing cells derived fromeither a multicellular plant or animal which allows for the cells toremain viable outside the original plant or animal. The term"downstream" identifies sequences proceeding farther in the direction ofexpression; for example, the coding region is downstream from theinitiation codon. The term "upstream" identifies sequences proceeding inthe opposite direction from expression; for example, the bacterialpromoter is upstream from the transcription unit, the initiation codonis upstream from the coding region. The term "microorganism" includesboth single cellular prokaryote and eukaryote organisms such asbacteria, actinomycetes and yeast. The term "operon" is a complete unitof gene expression and regulation, including structural genes, regulatorgenes and control elements in DNA recognized by regulator gene product.The term "plasmid" refers to an autonomous self-replicatingextrachromosomal circular DNA and includes both the expression andnonexpression types. Where a recombinant microorganism or cell cultureis described as hosting an expression plasmid the phrase "expressionplasmid" includes both extrachromosomal circular DNA and DNA that hasbeen incorporated into the host chromosome(s). Where a plasmid is beingmaintained by a host cell, the plasmid is either being stably replicatedby the cells during mitosis as an autonomous structure or as anincorporated portion of the host's genome. The term "promoter" is aregion of DNA involved in binding the RNA polymerase to initiatetranscription. The phrase "DNA sequence" refers to a single or doublestranded DNA molecule comprised of nucleotide bases, adenosine,thymidine, cytosine and guanosine. The phrase "essentially pure" refersto a composition of protein that contains no parainfluenza virus proteinother than the desired recombinant chimeric glycoprotein. Although theessentially pure proteins may be contaminated with low levels of hostcell constituents, the protein is devoid of contaminating structural andnon-structural viral protein produced by replicating parainfluenzaviruses. The phrase "suitable host" refers to a cell culture ormicroorganism that is compatible with a recombinant plasmid and willpermit the plasmid to replicate, to be incorporated into its genome orto be expressed.

This invention involves a series of molecular genetic manipulations thatcan be achieved in a variety of known ways. The manipulations can besummarized as obtaining a cDNA of the protein, the cloning andreplication of the cDNA in E. coli and the expression of the desiredcDNA in a suitable host. The following descriptions will detail thevarious methods available to express the protein and are followed byspecific examples of preferred methods.

Generally, the nomenclature and general laboratory procedures requiredin this invention can be found in Maniatis, et al., Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

All E. coli strains are grown on Luria broth (LB) with glucose, Difco'sAntibiotic Medium #2 and M9 medium supplemented with glucose andacid-hydrolyzed casein amino acids. Strains with resistance toantibiotics were maintained at the drug concentrations described inManiatis. Transformations were performed according to the methoddescribed by Rowekamp and Firtel, Dev. Biol., 79:409-418 (1980).

All enzymes were used according to the manufacturer's instructions.Transformants were analyzed by colony hybridization as described inGrunstein and Wallis, Methods in Enzymology, 68:379-388.

After hybridization, the probes are removed and saved, and the filtersare washed in 0.1% SDS, 0.2x SSC for a total of 3 hours with 5 changesof 400 ml each. Filters are thoroughly air dried, mounted, andautoradiographed using Kodak X-OMAT AR film and Dupont Cronex LighteningPlus intensifying screens for an appropriate time at -70° C.

For sequencing of plasmids, purified plasmid DNA is prepared accordingto the methods described in Maniatis. End-labeled DNA fragments areprepared and analyzed by the chemical sequencing methods of Maxam andGilbert with modifications described by Collins and Wertz, J. Virol.54:65-71 (1985).

Nucleotide sizes are given in either kilobases (Kb) or basepairs (bp).These are estimates derived from agarose gel electrophoresis.

The first step in obtaining expression of protein is to obtain the DNAsequence coding for the protein from cDNA clones. This sequence is thencloned into an expression plasmid which is capable of directingtranscription of the gene and allowing efficient translation of thetranscript. The library method for obtaining cDNA encoding proteins isdescribed generally in Maniatis, and specifically by Elango, et al., inHuman Parainfluenza Type 3 Virus Hemagglutinin-NeuraminidaseGlycoprotein: Nucleotide sequence of mRNA and Limited Amino AcidSequence of the Purified Protein, J. Virol. 57: 481-489 (1986) and bySpriggs, et al., in Fusion Glycoprotein of Human Parainfluenza VirusType 3: Nucleotide Sequence of the Gene, Direct Identification of theCleavage-Activation Site, and Comparison with Other Paramyxoviruses,Virology 152: 241-251 (1986).

Clones are prepared by inserting the cDNA into PstI cleaved pBR322 towhich homopolymer tracts of dGTP have been enzymatically added to the 3'ends at the cleavage site. Homopolymer tracts of dCTP are enzymaticallyadded to the 3' termini of the cDNA molecules according to the methodsdescribed by Maniatis. Ideally, 10-30 residues of dCTP or dGTP should beadded to maximize cloning efficiency. The cDNA and plasmid are annealedtogether and transformed into E. coli. The clones containing full lengthcDNA are detected by probes of labeled viral cDNA or oligonucleotidescomplementary to portions of the gene sequences, followed by restrictionenzyme analysis and DNA sequencing.

Oligonucleotides are chemically synthesized according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letters, 22(20):1859-1862 (1981) using anautomated synthesizer, as described in Needham-VanDevanter, et al.,Nucleic Acids Res., 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier, J. Chrom.,255:137-149 (1983).

The sequence of the synthetic oligonucleotides can be verified using thechemical degradation method of Maxam and Gilbert, Grossman and Moldave,eds., Academic Press, New York, Methods in Enzymology, 65:499-560(1980).

To obtain high level expression of a cloned gene in a prokaryoticsystem, it is essential to construct expression vectors which contain,at the minimum, a strong promoter to direct mRNA transcription, aribosome binding site for translational initiation, and a transcriptionterminator. Examples of regulatory regions suitable for this purpose arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky, Kelley, and Horn, J. Bacterial.,158:1018-1024 (1984) and the leftward promoter of phage lambda (P_(L))as described by Herskowitz and Hagen, Ann. Rev. Genet., 14:399-445(1980).

The proteins produced in E. coli will not fold properly due to thepresence of cysteine residues and to the lack of suitablepost-translational modifications. During purification from E. coli, theexpressed proteins must first be denatured and then renatured. This canbe accomplished by solubilizing the E. coli produced proteins inguanidine HCl and reducing all the cysteine residues withβ-mercaptoethanol. The protein is then renatured either by slow dialysisor by gel filtration, U.S. Pat. No. 4,511,503.

Detection of proteins is achieved by methods known in the art such asradioimmunoassays, or Western blotting techniques orimmunoprecipitation. Purification from E. coli can be achieved followingprocedures described in U.S. Pat. No. 4,511,503.

Expression of heterologous proteins in yeast is well known anddescribed. Methods in Yeast Genetics, Sherman, et al., Cold SpringHarbor Laboratory, (1982) is a well recognized work describing thevarious methods used to produce proteins in yeast.

For high level expression of a gene in yeast, it is essential to connectthe gene to a strong promoter system as in the prokaryote and to alsoprovide efficient transcription termination/polyadenylation sequencesfrom a yeast gene. Examples of useful promoters include GAL1,10,Johnston and Davis, Mol. and Cell. Biol., 4:1440-1448, 1984), ADH2,Russell, et al., J. Biol. Chem. 258:2674-2682, 1983), PHO5, EMBOJ.6:675-680, (1982), and MFα1. A multicopy plasmid with a selective markersuch as Lue-2, URA-3, Trp-1, or His-3 is also desirable. The MFα1promoter is preferred. The MFα1 promoter, in a host of the α mating-typeis constitutive, but is off in diploids or cells with the a mating-type.It can, however, be regulated by raising or lowering temperature inhosts which have a ts mutation at one of the SIR loci. The effect ofsuch a mutation at 35° C. on an α type cell is to turn on the normallysilent gene coding for the a mating-type. The expression of the silent amating-type gene, in turn, turns off the MFα1 promoter. Lowering thetemperature of growth to 27° C. reverses the whole process, i.e., turnsthe a mating-type off and turns the MFα1 on, Herskowitz and Oshima, TheMolecular Biology of the Yeast Saccharomyces, Strathern, Jones, andBroach, eds., Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.,181-209, (1982).

The polyadenylation sequences are provided by the 3'-end sequences ofany of the highly expressed genes, like ADH1, MFα1, or TPI, Alber andKawasaki, J. of Mol. and Appl. Genet. 1:419-434, (1982).

A number of yeast expression plasmids like YEp6, YEp13, YEp24 can beused as vectors. A gene of interest can be fused to any of the promotersmentioned above, and then ligated to the plasmids for expression invarious yeast hosts. These plasmids have been fully described in theliterature, Botstein, et al., Gene, 8:17-24, (1979); Broach, et al.,Gene, 8:121-133, (1979).

Two procedures are used in transforming yeast cells. In one case, yeastcells are first converted into protoplasts using zymolyase, lyticase orglusulase, followed by addition of DNA and polyethylene glycol (PEG).The PEG-treated protoplasts are then regenerated in a 3% agar mediumunder selective conditions. Details of this procedure are given in thepapers by Beggs, Nature (London), 275:104-109 (1978); and Hinnen, etal., Proc. Natl. Acad. Sci. USA, 75:1929-1933 (1978). The secondprocedure does not involve removal of the cell wall. Instead the cellsare treated with lithium-chloride or acetate and PEG and put onselective plates, Ito, et al., J. Bact., 153:163-168, (1983).

The cDNA can be ligated to various expression vectors for use intransforming host cell cultures. The vectors all contain gene sequencesto initiate transcription and translation of the proteins that arecompatible with the host cell to be transformed.

In addition, the vectors preferably contain a marker to provide aphenotypic trait for selection of transformed host cells such asdihydrofolate reductase or metallothionein. Additionally a replicatingvector might contain a replicon.

Insect or mammalian cell cultures are useful for the production ofproteins. Mammalian cell systems often will be in the form of monolayersof cells although mammalian cell suspensions may also be used.Illustrative examples of mammalian cell lines include VERO and HeLacells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7 or MDCKcell lines.

The vector which is used to transform the host cell preferably containsgene sequences to initiate the transcription and translation of theprotein's gene sequence. These sequences are referred to as expressioncontrol sequences. When the host cell is of mammalian or insect originillustrative useful expression control sequences are obtained from theSV-40 promoter, Science, 222, 524-527 (1983), the CMV I.E. promoter,Proc. Natl. Acad. Sci. 81:659-663 (1984), the metallothionein promoter,Nature, 296, 39-42, (1982) or the baculovirus polyhedrin promoter(insect cells), Virol., 131, 561-565 (1983). The plasmid for replicatingor integrating DNA material containing the expression control sequencesis cleaved using restriction enzymes and adjusted in size as necessaryor desirable an(i ligated with cDNA coding for proteins using methodswell known in the art.

When higher animal host cells are employed, polyadenylation ortranscription terminator sequences from known mammalian genes need to beincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene.Additionally gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovinepapillomavirus type-vectors, Saveria-Campo, "Bovine papillomavirus DNA:a eukaryotic cloning vector", DNA Cloning Vol. II--A practical approach,Glover, ed., IRL Press, Arlington, Va. 213-238 (1985).

The preferred expression vector useful for expressing proteins inChinese hamster ovary (CHO) cells is a shuttle vector pSVCOW7 whichreplicates in both CHO and E. coli cells utilizing ampicillin resistanceand dihydrofolate reductase genes as markers in E. coli and CHO cellsrespectively. Plasmid pSVCOW7 also provides the polyadenylation sequencefrom bovine growth hormone which is necessary for expression in CHOcells. Plasmid pSVCOW7 is cleaved and a viral promoter and cDNAsinserted.

The preferred expression vector useful in forming recombinantbaculovirus for expressing proteins in insect cells is pAc373, Smith, etal., Mol. Cell. Biol. 3:2156-2165 (1983). The plasmid replicates in E.coli cells utilizing ampicillin resistance, and provides the eukaryoticpromoter and polyadenylation signal from the baculovirus polyhedrin genefor expression of genes. Plasmid pAc373 is cleaved and a cDNA isinserted adjacent to the promoter. This new plasmid is cotransfectedwith baculovirus (Autograpa californica nuclear polyhedrosis virus) DNAinto insect cells by calcium phosphate precipitation. Recombinantbaculovirus in which the pAc373 polyhedrin gene containing a cDNA hasreplaced the resident viral polyhedrin gene by homologous recombinationis detected by dot blot hybridization using ³² P-labeled cDNA as aprobe, Summers and Smith, A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures, Texas A & M University, CollegeStation, Tex., 29-30 (1986). Insect cells infected with recombinantbaculovirus may also be differentiated by their occlusion-negativemorphology since the insertion of the cDNA into the polyhedrin geneprevents the synthesis of this occlusion-forming protein.

The preferred expression vector used in conjunction with bovinepapilloma virus (BPV) for expressing proteins is pTFW9 (Plasmid pTWF9was deposited in accordance with the Budapest Treaty. Plasmid pTFW9 ismaintained in an E. coli host and has been deposited with the NorthernRegional Research Center, Peoria, Ill., U.S.A. on Nov. 17, 1986 andassigned Accession Number NRRL B-18141.) The plasmid replicates in E.coli utilizing ampicillin resistance, and provides the mousemetallothionein promoter and SV40 polyadenylation signal for expressionof genes. Plasmid pTFW9 is cleaved and a cDNA is inserted adjacent tothe promoter. This new plasmid is then cleaved to allow insertion ofBPV. The recombinant plasmid is transfected into animal cells by calciumphosphate precipitation and foci of transformed cells are selected.

The host cells are competent or rendered competent for transfection byvarious means. There are several well-known methods of introducing DNAinto animal cells. These include: calcium phosphate precipitation,fusion of the recipient cells with bacterial protoplasts containing theDNA, treatment of the recipient cells with liposomes containing the DNA,and microinjection of the DNA directly into the cells. The transfectedcells are cultured by means well known in the art, Biochemical Methodsin Cell Culture and Virology, Kuchier, Dowden, Hutchinson and Ross,Inc., (1977). Recombinant glycoproteins expressed in one of the aboveeukaryotic expression systems are isolated from cell suspensions createdby disruption of the host cell system by well known mechanical orenzymatic means. Proteins which are designed to be secreted from thecells are isolated from the media without disruption of the cells. Forpurification of glycoproteins it is helpful to first apply thecytoplasmic fraction to a lentil lectin column which will specificallybind glycoproteins. The eluted glycoproteins are then applied to anaffinity column containing antibody.

A typical glycoprotein can be divided into three regions. At the aminoterminal end is a hydrophobic region called the signal sequence. Thissequence of amino acids signals the transport of the glycoprotein to thecell membrane. Following transport the signal sequence is removed bycleavage. Downstream from the signal sequence is the extracellulardomain of the mature glycoprotein. This is the immunogenic portion ofthe glycoprotein since it is accessible to antibodies. At the carboxyterminal end of the glycoprotein is the hydrophobic anchor region whichcauses the glycoprotein to be retained in the cell membrane. The PIV3 Fis a typical glycoprotein in that it contains an amino terminal signalsequence and carboxy terminal anchor sequence, Spriggs, et al., Virology152:241-25, (1986). However, the PIV3 HN glycoprotein is unusual sinceits amino terminal end acts as both a signal and anchor region, Elango,et al., J. Virol. 57:481-489, (1986).

A glycoprotein may be designed to be secreted from cells into thesurrounding media. This is accomplished by causing the early terminationof the glycoprotein before translation of the anchor region, Lasky, etal., Biotechnology, 2:527-532 (1984). Early termination may beaccomplished by inserting a universal translational terminatoroligonucleotide into an appropriate site in the gene's DNA. Theseoligonucleotides are commercially available. Early termination may alsobe accomplished by altering the reading frame, thus generating atranslational termination codon.

The chimeric glycoprotein described below consists of the signal andextracellular domains of PIV3 F linked to the extracellular domain ofPIV3 HN, and will be referred to as FHN. When properly placed in aeukaryotic expression vector, the FHN gene described above is designedto express a chimeric glycoprotein which would be transported to thecell's surface and secreted into the media.

The majority of the cytoplasmic domain of the PIV3 HN protein iscontained within the coding region spanned by the DraI (nucleotideposition 452 of the protein coding region) and PstI (nucleotide position1709 of the protein coding region) restriction enzyme sites. Thissequence does not code for the signal/anchor region of the glycoprotein.The majority of the cytoplasmic domain of the PIV3 F protein iscontained within the coding region prior to the XbaI (nucleotideposition 1398 of the protein coding region) restriction enzyme site.This sequence codes for the signal region and the majority of theantigenic region, but not the anchor region of the F glycoprotein.

To insert the HN glycoprotein sequence into the F glycoprotein of PIV3,the HN gene is digested with PstI and the end is made blunt with T4 DNApolymerase. An XbaI linker (New England Biolabs) with the sequence##STR1## is ligated to the end. The gene is separated from residuallinker by agarose gel electrophoresis. The above linker contains an inphase translation termination signal to stop protein synthesis. The HNgene is then digested with DraI and a XbaI linker (New England Biolabs)with the sequence ##STR2## is ligated to the end. This linker does notcontain an in phase translation termination signal and will allow readthrough of the protein from the F to the HN sequences. The HN genefragment (1.3 Kb) containing the linkers is digested with XbaI andseparated from residual linkers by agarose gel electrophoresis. The PIV3F gene is digested with XbaI and dephosphorylated with bacterialalkaline phosphatase. The 1.3 Kb HN fragment is then ligated into the Fgene at the XbaI site and transformed into E. coli HB101. A clonecontaining the chimeric glycoprotein gene is isolated and the junctionsbetween the F and HN DNA sequences are verified correct by Maxam-Gilbertsequencing. The PIV3 chimeric glycoprotein gene can be placed in anappropriate expression vector.

The above restriction enzyme sites were chosen because they allow forthe expression of a large proportion of the relevant regions of the Fand HN glycoproteins. However, other portions of the glycoproteins couldbe expressed by choosing other restriction enzyme sites within the F andHN coding sequences for the fusion of these genes. For instance, therestriction enzymes HaeIII, KpnI, or NlaIV could be used to cleave atthe 5' end of the HN gene. The restriction enzymes BalI, BglII, orHaeIII could be used to cleave at the 3' end of the HN gene. The enzymescould be used in any combination of two with one enzyme being from eachgroup to give immunogenic protein fragments. For the gpF gene, theBglII, HaeIII, NsiI, or XhoII restriction enzymes could be used in placeof XbaI. Linker oligonucleotides could be added to correct the readingframe in the junction regions. Two oligonucleotides which would correctthe two possible frame shifts are the SalI linkers ##STR3## which arecommercially available. Also when an anchor region is desired in theglycoprotein, a linker oligonucleotide is added a the second junction toallow synthesis of the gpF anchor region. Alternative strategies couldbe designed for the expression of a FHN chimeric protein by insertion ordeletion of various sequences. The major criterion for the protein isthe retention of a signal sequence and the immunologically importantregions of the two glycoproteins.

Insertion of FHN gene into CHO, BPV, or baculovirus expression vectorsis as already described.

The FHN chimeric glycoprotein offers advantages over expression of theindividual glycoproteins. Since FHN is a single protein, it requireshalf the labor and reagents for purification compared to the separate Fand HN glycoproteins. Also, the FHN chimeric glycoprotein is secretedinto the media for ease of purification. The F glycoprotein can beengineered as a secreted glycoprotein by truncation prior to the anchorregion sequences. However, the PIV3 HN glycoprotein contains asignal/anchor region at its amino terminal end. Therefore, truncation ofthis glycoprotein will not generate a secreted form. The signal/anchorregion could be replaced with a signal region from a foreignglycoprotein, but this would introduce foreign protein sequences intothe potential vaccine.

Conventions used to represent plasmids and fragments in Charts 1-6, aremeant to be synonymous with conventional circular representations ofplasmids and their fragments. Unlike the circular figures, the singleline figures on the charts represent both circular and lineardouble-stranded DNA with initiation or transcription occurring from leftto right (5' to 3'). Asterisks (*) represent the bridging of nucleotidesto complete the circular form of the plasmids. Fragments do not haveasterisk marks because they are linear pieces of double-stranded DNA.Endonuclease restriction sites are indicated above the line. Genemarkers are indicated below the line. The relative spacing betweenmarkers do not indicate actual distances but are only meant to indicatetheir relative positions on the illustrated DNA sequence.

Example 1 Removing the G-C tails from the F glycoprotein gene--Chart 1

In order to obtain maximum expression of the F glycoprotein, the G-Cnucleotides which are used to insert the cDNA into the plasmid pBR322must be removed from the ends of the cDNA. In order to convenientlyinsert the FHN cDNA into the preferred expression vector for CHO cells(pSVCOW7, described below), or the preferred expression vector forbaculovirus (pAc373, described below), it is necessary to supply a BamHIsite upstream from the protein coding sequence. Methods for thesynthesis of the cDNA clones containing the entire sequence for the Fglycoprotein have been described. Spriggs, et al., Virology 152:241-251, (1986).

The cDNA containing the intact PIV3 F gene (pGPF1) is digested withBstXI and NdeI. BstXI cleaves the F gene at position 39 relative to thegene's initiation codon, and NdeI cleaves at position 1599.Oligonucleotide I is ligated to the BstXI cleavage site andoligonucleotide 2 is ligated to the NdeI cleavage site. OligonucleotideI contains the DNA sequences from 10 bases prior to the coding region tothe BstXI cleavage site in the coding region of the F gene (-10 to +39),and has a BamHI site on the 5' end of the oligonucleotide.Oligonucleotide 2 contains the DNA sequences from the NdeI cleavage siteto the termination codon of the F gene (+1599 to +1620). At the 3' endof oligonucleotide 2 is a NruI restriction enzyme site followed by aBamHI restriction enzyme site.

Following ligation of the oligonucleotides, the DNA is digested withBamHI and the F gene (fragment 1, 1.6 Kb) is gel purified. Fragment I isligated into plasmid pBR322 (Pharmacia) which has been digested withBamHI and dephosphorylated with bacterial alkaline phosphatase. Theplasmid (pGPF2) is transformed into E. coli HB101. The newly synthesizedregions of pGPF2 are sequenced by the Maxam-Gilbert procedure to verifyaccurate synthesis and ligation. ##STR4##

Example 2 Construction of a PIV3 Chimeric FHN Gene--Chart 2

A. Preparation of the PIV3 HN glycoprotein gene. Clones containing theentire coding region of the PIV3 HN gene and methods for isolating suchclones have been described. Elango, et al., J. Virol. 57:481-489,(1986). A cDNA clone containing the PIV3 HN gene (pGPHN1) is digestedwith PstI. This enzyme cleaves toward the 3' end of the HN gene(nucleotide +1714). The ends of the fragment are made blunt with T4 DNApolymerase and then dephosphorylated with bacterial alkalinephosphatase. An XbaI linker (New England Biolabs; linker 1) with thesequence ##STR5## is ligated to the end. The cDNA is separated fromresidual linker by electrophoresis in a 1.2% agarose gel. The 1.7 Kbfragment (fragment 2) containing the HN gene is excised from the gel andthe DNA is purified from the agarose. The above linker contains an inphase translation termination signal to stop protein synthesis. The HNgene is then digested with DraI. This enzyme cleaves 3' to thesignal/anchor encoding region of the HN gene (nucleotide +452). A XbaIlinker (New England Biolabs; linker 2) with the sequence ##STR6## isligated to the end. This linker does not contain an in phase translationtermination signal. The DNA is digested with XbaI and separated fromresidual linkers by electrophoresis in a 1.2% agarose gel. The 1.3 Kbfragment containing the relevant region of the HN gene is excised fromthe gel and the DNA is purified from the agarose.

B. Insertion of the HN cDNA into the PIV3 F glycoprotein gene.

Plasmid pGPF2 is digested with XbaI and dephosphorylated with bacterialalkaline phosphatase. The 1.3 Kb fragment is ligated into the XbaI siteto yield the chimeric FHN gene (pGPFHN1). The plasmid is transformedinto E. coli HB101. Clones are isolated and selected from the correctorientation of the HN cDNA within the F gene by digestion with BamHI andPvuII which will generate fragments of approximately 2.5 Kb and 350 bpwithin the FHN gene. The incorrect orientation of the HN fragment willyield fragments of approximately 1.5 Kb and 1.3 Kb upon digestion withBamHI and PvuII. The junction regions of a properly orientated clone aresequenced by the Maxam-Gilbert technique to verify proper ligation ofthe HN fragment.

Example 3 Using DNA oligonucleotides to generate genes coding forchimeric FHN glycoproteins of various lengths--Chart 3

Genes coding for chimeric FHN glycoproteins containing various regionsof the F and HN glycoproteins can be generated using a combination ofrestriction enzymes and oligonucleotides. This procedure allows the Fand HN glycoproteins to be linked at any desirable point of their aminoacid backbone, permitting incorporation or removal of regions likely tocontain epitopes which will be recognized by the host immune system.Individual amino acids may also be changed if so desired.Oligonucleotides are synthesized corresponding to the DNA sequence fromthe point of desired linkage to a convenient restriction enzyme site.The glycoprotein gene is digested with that restriction enzyme and theoligonucleotide is ligated to the gene at the restriction enzyme site togenerate a DNA fragment of the desired length. The oligonucleotides aresynthesized with ends compatible with the restriction enzyme sites forease of ligation.

A. Insertion of Glycoprotein HN cDNA into the F Glycoprotein Gene.

Clone pGPHN1 is digested with PstI and DraI. The 1.3 Kb fragmentrepresenting the cDNA region from nucleotide position 452 to 1714(fragment 4) is gel purified. Oligonucleotides representing adjoiningregions of the HN cDNA are then ligated to each end of fragment 4. TheDNA sequences in these oligonucleotides may code for additional epitopesfound on the HN glycoprotein. The individual oligonucleotides weredesigned to incorporate regions which may contain unique epitopes. Theoligonucleotide ligated to the 5' end of the HN cDNA may consist ofeither oligonucleotide 3 (cDNA nucleotides 395 to 452), oligonucleotides3-4 (cDNA nucleotides 335 to 452), oligonucleotides 3-4-5 (cDNAnucleotides 275 to 452), oligonucleotides 3-4-5-6 (cDNA nucleotides 218to 452), or oligonucleotides 3-4-5-6-7 (cDNA nucleotides 162 to 452).Parentheses enclose nucleotides which would be included only in theterminal oligonucleotide. For instance, the enclosed nucleotides wouldnot be included on oligonucleotide 5 if oligonucleotide 6 were to beadded. These enclosed nucleotides code for a XbaI site. The enclosednucleotides are not included when an additional oligonucleotide(s) is tobe added in order to allow ligation between the compatible ends of theoligonucleotides. For instance, the 5' end of the oligonucleotide 3 iscompatible with the 3' end of oligonucleotide 4 when the nucleotidesenclosed by parentheses are not included in oligonucleotide 3.Oligonucleotide 8 is ligated to the 3' end of the HN gene fragment. The5' end of this oligonucleotide is compatible with the 3' end of fragment4. The 3' end of this oligonucleotide contains an in phase translationtermination signal followed by a XbaI restriction enzyme site.

Following ligation of the oligonucleotides to the HN cDNA fragment, theDNA is digested with XbaI and the enlarged HN cDNA fragment (fragment 5)is gel purified. The new HN cDNA fragment is then ligated into XbaIdigested pGPF2. The DNA is transformed into E. coil HB101 and a clonecontaining the HN gene in the correct orientation within the F gene isisolated (pGPFHN2). Orientation is determined by digestion withappropriate restriction enzymes. The newly synthesized regions of thechimeric gene are verified correct by Maxam-Gilbert sequencing. Theclone may then be placed in various expression vectors as describedbelow. ##STR7##

Example 4 Construction of a PIV3 chimeric FHN glycoprotein genecontaining an anchor region--Chart 4

Examples 2 and 3 illustrate the synthesis of genes coding for chimericFHN glycoproteins which do not contain anchor regions and will thereforebe secreted into the medium of expressing cells. A gene coding for achimeric FHN glycoprotein containing an anchor region can besynthesized. The anchor region would cause the retention of the chimericglycoprotein in the cellular membranes in a manner similar to most viralglycoproteins. The anchor region may be on the carboxy-terminal end ofthe glycoprotein so that the immunogenic regions of the chimericmolecule from both the F and HN glycoproteins would protrude into theextracellular fluid. The gene described below will code for a chimericglycoprotein consisting of the extracellular region of PIV3 F, theextracellular region of PIV3 HN, and the anchor region of PIV3 F in theabove order from amino-terminus to carboxy-terminus.

A. Insertion of the HN cDNA fragment into the PIV3 F glycoprotein gene.

The clone pGPHN1 is digested with DraI and PstI. Oligonucleotide 9 isfirst ligated to the DNA fragment (oligonucleotide is compatible withDraI site). Oligonucleotide 10 is then ligated to the DNA fragment(compatible with PstI site). Both oligonucleotides contain XbaIrestriction enzyme sites. ##STR8## Following ligation, the DNA isdigested with XbaI and the 1.3 Kb fragment of the HN cDNA (fragment 6)is gel purified. Fragment 6 is then ligated into XbaI digested pGPF2.The DNA is transformed into E. coli HB101. Clones are isolated andselected from the correct orientation as described in Example 2. Thejunction regions of a properly orientated clone are then verifiedcorrect by Maxam-Gilbert sequencing. This clone (pGPFHN3) may be placedin various expression vectors as described below.

Example 5 Construction of a PIV3 chimeric HNF glycoprotein gene

A portion of the extracellular region of the PIV3 F glycoprotein may beplaced at the carboxy-terminal end of the HN glycoprotein. This chimericglycoprotein would consist of the signal/anchor region from theamino-terminus of HN, the majority of the extracellular region of HN,and a portion of the extracellular region of F in the above order fromamino-terminus to carboxy-terminus.

A. Preparation of the PIV3 HN glycoprotein gene--Chart 5.

To prepare clone pGPHN1 for expression, the G-C tails used in cDNAcloning must be removed and compatible restriction enzyme sites placedon its ends. Clone pGPHN1 is digested with HhpI. HphI cleaves atposition 75 on the cDNA gene coding sequence. The followingoligonucleotide is then ligated to the cDNA fragment: ##STR9##Oligonucleotide 11 will ligate to the HphI site and generate a BamHIrestriction enzyme site on the 5' end of the cDNA fragment. Followingligation of oligonucleotide 11, the DNA is digested with BamHI and PstI(PstI cleaves at nucleotide position 1714 in the HN gene). The DNA iselectrophoresed in a 1.2% agarose gel. The 1.7 Kb HN cDNA fragment(fragment 7) is excised from the gel and the DNA is purified from theagarose. Fragment 7 is then ligated into pUC19 which has been digestedwith BamHI and PstI to yield pGPHN2. The plasmid is transformed into E.coli HB101 and plasmid DNA is isolated.

B. Insertion of an F cDNA fragment into the PIV3 HN glycoproteingene--Chart 6.

The clone pGPF1 is digested with BstEII and XbaI. BstEII cleaves atposition 190 and XbaI at position 1398 on the F cDNA gene sequence. Thefollowing oligonucleotides are then ligated to the cDNA fragment.##STR10## Oligonucleotide 12 will ligate to the BstEII site and willgenerate a PstI restriction enzyme site on the 5' end of the cDNAfragment. Oligonucleotide 13 will ligate to the XbaI site and willgenerate a translational termination codon, a NruI restriction enzymesite, a BamHI restriction enzyme site, and a PstI restriction enzymesite in the indicated order (5' to 3') on the 3' end of the cDNAfragment. The DNA is then digested with PstI and the 1.2 Kb F cDNAfragment (fragment 8) is gel purified. Fragment 8 is then ligated intopGPHN2 which has been digested with PstI. The plasmid is transformedinto E. coli HB101. Clones are isolated and selected from the correctorientation of the F cDNA within the HN gene by digestion with BamHI andNruI which will generate a 2.9 Kb fragment. The incorrect orientationwill generate a 1.7 Kb fragment. The junction regions of a properlyorientated clone are then verified correct by Maxam-Gilbert sequencing.This clone (pGPHNF1) may be placed in various expression vectors asdescribed below.

Example 6 Expression of the Chimeric FHN Glycoprotein of PIV3 in CHOCells

A. Construction of pSVCOW7

The starting plasmid pSV2dhfr (available from the American Type CultureCollection or prepared according to the procedure of S. Subramani, etal., "Expression of the Mouse Dihydrofolate Reductase ComplementaryDeoxyribonucleic Acid in Simian Virus 40", Molecular and CellularBiology 2:854-864 (September 1981) is digested with BamHI and EcoRI toyield the 5.0 Kb fragment (fragment 9) containing the ampicillinresistance gene, the SV40 origin, and the dhfr gene. The second portionof pSVCOW7 is obtained from plasmid p GH2R2 which is digested with thesame restriction endonucleases used to cleave pSV2dhfr to obtain the 2.1Kb fragment (fragment 10) containing the 3' end of genomic bovine growthhormone gene, i.e., BGH gDNA. Plasmid p GH2R2 is publicly available froman E. coli HB101 host, deposited with the Northern Regional ResearchLaboratories in Peoria, Ill. (NRRL B-15154). Fragments 9 and 10 areligated to yield pSVCOW7 (7.1 Kb).

B. Construction of pGPFHN-IE-PA

The genes constructed in Examples 2-5 may be used for expression of achimeric glycoprotein in CHO cells. The plasmid pGPFHN-1 will be used inthe following example. The other chimeric genes are treated as describedfor pGPFHN-1 except when otherwise indicated. The assembly ofpGPFHN-IE-PA is accomplished in two steps. First the gpFHN cDNA frompGPFHN1 is inserted into pSVCOW7 yielding pGPFHN-PA and then theimmediate early promoter of cytomegalovirus is inserted to initiatetranscription of the PIV3-like proteins yielding pGPFHN-IEPA.

STEP 1. Plasmid pSVCOW7 is cut with EcoRI and PuvII and fragment 11 (600bp) containing the polyadenylation sequence of bovine growth hormoneextending from the PvuII site in the 3' most exon of the BGH gene, tothe EcoRI site downstream from the 3' end is isolated. For a completediscussion of the BGH polyadenylation sequence see the followingreferences: (1) European patent application 01 12012, published on 27Jun. 1984 wherein the identification and characterization of BGH genomicDNA is disclosed; (2) Woychik, R. P. et al., "Requirement for the 3'Flanking Region of the Bovine Growth Hormone Gene for AccuratePolyadenylation", Proc. Natl. Acad. Sci. USA 81:3944-3948 (July 1984);and, D. R. Higgs, et al., Nature 306:398-400 (24 Nov. 1983) andreferences cited therein disclosing that the nucleotide sequence AATAAAcharacterizes the polyadenylation signal at a location 11 to 30nucleotides upstream (towards the 5' end) from the 3' end of the BGHgene.

A second sample of pSVCOW7 is cut with EcoRI and BamHI to yield fragment12 (5.8 Kb). Fragment 12 can be alternatively derived from theEcoRI/BamHI fragment from parent plasmid pSV2dhfr available fromBethesda Research Laboratories. Fragment 12 contains the origin ofreplication from pBR322 and an ampicillin resistance gene expressed inE. coli which allows for the selection of the plasmid in E. coli. Thefragment also contains the mouse dihydrofolate reductase cDNA in aconstruction that allows expression in mammalian cells. Subramani, etal., Mol. Cell. Biol. 1:854-864 (1981).

Plasmid pGPFHN1 is cut with BamHI and NruI to yield fragment 13 (2.7 Kb)which is gel isolated. The BamHI site is just upstream from the cDNAcoding for the 5' untranslated sequences of the FHN MRNA, and the NruIsite is a few bases downstream from the translation termination codon.

Fragments 11, 12 and 13 are ligated to form pGPFHN-PA (9.1 Kb) which isa replication vector capable of shuttling between E. coli and CHO cells.Plasmid pGPFHN-PA is transformed into E. coli.

STEP 2. In step 2, pGPFHN-PA is converted into expression plasmidpGPFHN-IE-PA by inserting the immediate early gene promoter from humancytomegalovirus (CMV I.E. promoter). The CMV I.E. promoter is obtainedfrom the PstI digestion of the CMV genome. The restriction endonucleasecleavage maps of the region of the human cytomegalovirus (CMV) genomecontaining the major immediate early gene (CMV I.E.) have been describedin detail Stinski, et al., J. Virol. 46:1-14, 1983; Stenberg, et al., J.Virol. 49:190-199, 1984; and, Thomsen, et al., Proc. Natl. Acad. Sci.USA, 81:659-663, 1984.

The Stinski and Thomsen references describe a 2.0 kilobase PstI fragmentwhich contains the promoter for the major immediate early gene. Whenthis 2.0 Kb PstI fragment is isolated and digested with Sau3AI, a 760basepair fragment is obtained among the products. This 760 base pairfragment can be distinguished from the other products by its size andthe presence of a SacI cleavage site and a BalI cleavage site within thefragment. Because of its convenient identification, utilization of thisSau3AI fragment is the preferred method of use of the CMV I.E. promoteras described in the present specification.

Plasmid pGPFHN-PA is cleaved with BamHI, and a Sau3AI fragmentcontaining the CMV immediate early promoter is ligated into thecompatible BamHI site. Plasmids containing the CMV promoter fragment inan orientation such that transcription from the promoter wouldsynthesize an MRNA for a PIV3-like protein are identified by cleavage ofthe plasmids with SacI. The resulting plasmid is designated pGPFHN-IE-PAhaving the CMV I.E. promoter at the 5'-end of the cDNA and the BGHpolyadenylation signal on its 3'-end. The plasmid is maintained in E.coli until transfection into CHO cells.

C. Transfection and Culturing of CHO Cells.

Plasmid pGPFHN-IE-PA is transfected into Chinese hamster ovary (CHO)cells deficient in dihydrofolate reductase(dhfr) using the calciumphosphate method for transfection of DNA into cells which is describedin detail by Graham, et al., Introduction of Macromolecules into ViableMammalian Cells, Alan R. Liss Inc., N.Y., 1980, pp. 3-25. The cell lineused is the mutant DXB-11 originally available from L. Chasin, ofColumbia University and completely described in Proc. Natl. Acad. Sci.USA 77:4216-4220 (1980). The above methods for transfection relies onthe fact that cells which incorporate the transfected plasmids are nolonger dhfr deficient and will grow in Dulbeeco's modified Eagle'smedium plus proline.

If the chimeric glycoprotein does not contain an anchor region, thensupernatant from CHO cells expressing secreted chimeric FHN protein isclarified by low speed centrifugation. The supernatant is applied to aconconavalin A or lentil lectin column. The glycoprotein is eluted afterextensive washing with a linear gradient of a α-D-methylglucoside (0-0.5M) in the above buffer. The eluted glycoprotein is dialyzed against PBScontaining 0.1% Triton X-100 and applied to an affinity column. Theaffinity column is composed of either polyclonal or monoclonalantibodies directed against PIV3 linked to Sepharose 4B beads(Pharmacia, Piscataway, N.J.) by known techniques. The column is washedin dialysis buffer and the PIV3 FHN glycoprotein is eluted with PBScontaining 0.1M glycine (pH 2.5) and 0.1% Triton X-100. The glycoproteinis dialyzed against saline and checked for purity by electrophoresis ona SDS-PAGE gel.

If the chimeric glycoprotein contains an anchor region, then the CHOcells expressing the glycoprotein are washed in phosphate bufferedsaline (PBS) and then lysed in PBS containing 1.0% Triton X-100 and 1.0%sodium deoxycholate. After pelleting the nuclei, the cytoplasmic extractis applied to a conconavalin A column and purified as described abovefor secreted glycoproteins.

Example 7 The Expression of PIV3 GPFHN Using Bovine Papilloma Virus(BPV)

A. The construction of a cloning vector containing a nontranscribableexpression cassette suitable for replication in E. coli

The constructions of pTFW8 and pTFW9 offer a convenient startingmaterial for expressing PIV3 proteins using BPV. The transcriptionterminator of the deposited plasmid prevents the expression of PIV3proteins and must be removed in a single step excision and ligation.

1. Construction of pTFW8

Plasmid pdBPV-MMTneo (342-12) described in Mol. and Cell Biol., Vol 3(No. 11):2110-2115 (1983) and obtained from Peter Howley of the NationalCancer Institute, Bethesda, Md., USA. Plasmid pdBPV-MMT neo (342-12)consists of three parts: a complete BPV-1 genome (100%) opened at theunique BamHI site; pML2 (a "poison-minus" derivative of pBR322); and atranscriptional cassette composed of the murine metallothionein I genepromoter, the neomycin phosphotransferase 11 gene of Tn5, and the simianvirus 40 early-region transcriptional processing signals. PlasmidpdBPV-MMT neo (342-12) is first digested with BamHI to remove the BPVsequences which were isolated and stored for later insertion. Theremaining fragment is religated using T4 ligase to form pMMpro.nptII(6.7 Kb). Removal of the BPV genome facilitates later geneticmanipulations by creating unique restriction sites in the remainingplasmid. After the recombinations are complete, the BPV genome isreplaced.

Plasmid pMMpro.nptII is digested with BglII and a synthetic DNA fragment14 containing unique restriction sites is inserted and ligated using T4ligase to yield pTFW8 (6.7 Kb). Plasmid pTFW8 is identical topMMpro.nptII except for the insertion of unique restriction sitesbetween the murine metallothionein I gene promoter and the neomycinresistance gene.

2. Construction of pTWF9

Plasmid pTWF9 contains the transcription terminator T_(I) from phagelambda inserted between the metallothionein I gene promoter and theneomycin resistance gene. The transcription terminator can be obtainedfrom Donald Court of the National Cancer Institute in Bethesda, Md. USA.The transcription terminator is supplied in pKG1800sib3 which is thesame as pUS6 as described in Gene, 28:343-350 (1984), except that t_(I)carries the sib3 mutation as described in Guarneros, et al., PNAS,79:238-242 (1982). During the normal infection process of phage lambda,the t_(I) terminator functions in the inhibition of bacteriophage intgene expression from P_(L) and in the termination of int genetranscription originating from P_(I). The terminator is excised frompKG1800sib3 using AluI and PvuI as fragment 15 (1.2 Kb), which is gelisolated and XhoI linkers are placed on either end of the fragment.

The linkers are available from New England Biolabs, Beverly, Ma., U.S.A.The terminator fragment bounded by XhoI complementary ends is theninserted into pTWF8 which has been previously digested with XhoI. Thefragments are then ligated using T4 DNA ligase to yield pTWF9 (7.9 Kb).Plasmid pTWF9 was deposited in accordance with the Budapest Treaty.Plasmid pTFW9 is maintained in an E. coli host and has been depositedwith the Northern Regional Research Center, Peoria, Ill., U.S.A. on Nov.17, 1986 and assigned Accession Number NRRL B-18141.

B. The construction of pTFW/GPFHN

The genes constructed in Examples 2-5 may be used for expression of achimeric glycoprotein using BPV. The plasmid pGPFHN-1 will be used inthis example. The other chimeric genes are treated as described forpGPFHN-1 except when otherwise indicated. To construct pTFW/GPFHN,pGPFHN1 is digested with BamHI. Its ends are made flush with Klenowenzyme and synthetic BglII linkers (New England Biolabs) are ligated tothe ends of the clone. The DNA is digested with BglII and designatedfragment 16 (2.7 Kb). Fragment 16 containing the gpFHN gene (2.7 Kb) isthen isolated from a gel. The purified fragment is ligated into pTFW9which has been digested with BglII to yield pTFW/GPFHN (10.6 Kb).

C. Conversion of pTFW/GPFHN into a eukaryote expression vector

Plasmid pTFW/GPFHN is converted into a eukaryote expression vector byreinserting the 100% complete BPV-1 genome excised with BamHI. PlasmidpTFW/GPFHN is cut with BamHI and the BPV-1 intact genome, a 7.9 Kbfragment is inserted to yield pTFW/GPFHN/BPV* (18.5 Kb) which isreplicated in E. coli until production of glycoprotein FHN by eukaryoticcells is desired.

D. Expression of gpFHN in murine C127 cells

Prior to transfection into murine C127 cells, pTFW/GPFHN/BPV* isdigested with XhoI to excise the T₁ terminator and religated with T4 DNAligase. The resulting plasmid pTFW/GPFHN/BPV (17.4 Kb) will now directthe expression of high levels of gpFHN which is secreted into theculture media. The C127 cells are available from the American TypeCulture Collection and grown in Dulbecco's modified minimal essentialmedia containing 10% fetal calf serum. The levels of gpFHN proteins inthe media of the C127 cells are determined by Western blot experimentswith anti-PIV3 antibody and 125_(I) -labeled protein A.

PIV3 gpFHN is purified from the culture media or cells as described inExample 6.

Example 8 The Expression of PIV3 GPFHN Using Baculovirus Virus

The following example relates to the expression of glycoprotein FHN ininsect cell cultures. All procedures are detailed in Summers, M. D. andSmith, G. E., A Manual for Baculovirus Vectors and Insect Cell CultureProcedures published by the College of Agriculture, Texas AgriculturalExperiment Station, Texas Agricultural Extension Service, CollegeStation, Tex., 1986. The starting plasmid pAc373 (7.1 Kb) is a generalbaculovirus expression vector having a unique BamHI site immediatelydownstream from the polyhedron promoter for Autographs californicanuclear polyhedrosis virus (AcNPV). The polyhedron protein is a matrixprotein that is nonessential for viral infection and replication invitro. The 5plasmid is available from Professor Max Summers of theDepartment of Entomology, Texas A & M University, College Station, Tex.77843 and is fully described in Molecular and Cell. Biology,3(12):2156-2165 (1983).

A. Construction of pAcGPFHN

The genes constructed in Examples 2-6 may be used for expression of achimeric glycoprotein using baculovirus. The plasmid pGPFHN1 will beused in this example. The other chimeric genes are treated as describedfor pGPFHN1 except when otherwise indicated. Plasmid pGPFHN1 is digestedwith BamHI and fragment 17 (2.7 Kb) containing the gpFHN gene isisolated from a gel. The purified fragment is ligated into pAc373 whichhas been digested with BamHI.

B. Transfection and culturing of S. Frugiperda

The gpFHN cDNA insert of pAcGPFHN is recombined with native AcNPV DNA bycotransfection in S. frugiperda. S. Frugiperda (SF9; ATCC CRL 171 1) arecultured in Grace Media (Gibco Lab. Livonia, Mich. 48150), 10% fetalcalf serum and supplemented with Difco Lactalbumin hydrolysolate andyeastolate. The cells are cotransfected with AcNPV DNA and pAcGPFHN at 1μg/ml and 2 μg/ml respectively. Resulting virus particles are obtainedby collecting the media and removing cellular material by low speedcentrifugation. The virus containing-media is then used to infect S.frugiperda. Subsequent infection of S. frugiperda using these viralparticles which include both native viral DNA and DNA recombined withthe cDNA coding for glycoprotein FHN will result in some cellsexpressing the PIV3 protein instead of the polyhedron protein.Purification of recombinant virus is accomplished by a series of limiteddilution platings in 96-well tissue culture plates containing S.frugiperda cells. Wells containing recombinant virus are detected by dotblot hybridization using pGPFHN1 which has been labeled with ³² p-dCTPby nick translation as a probe. Once sufficiently pure, the recombinantvirus is detected by its unique occlusion-negative plaque morphology.PIV3 protein synthesized in recombinant baculovirus infected cells isdetected by Western blot experiments with anti-PIV3 antibody and ¹²⁵I-labeled protein A (Amersham Corp.).

The PIV3 protein is purified from the culture media or cells asdescribed in Example 6.

Example 9 Preparation of a Vaccine

The immunogen can be prepared in vaccine dose form by well-knownprocedures. The vaccine can be administered intramuscularly,subcutaneously or intranasally. For parenteral administration, such asintramuscular injection, the immunogen may be combined with a suitablecarrier, for example, it may be administered in water, saline orbuffered vehicles with or without various adjuvants or immunomodulatingagents such as aluminum hydroxide, aluminum phosphate, aluminumpotassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon,water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide,bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacteriumacnes), Bordetella pertussis, polyribonucleotides, sodium alginate,lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,DEAE-dextran, blocked copolymers, ISCOMS or other synthetic adjuvants.Such adjuvants are available commercially from various sources, forexample, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminum hydroxide can be present in an amount of about 0.5 % of thevaccine mixture (Al₂ O₃ basis). On a per dose basis, the concentrationof the immunogen can range from about 0.015 μg to about 1.5 mg perkilogram per patient body weight. A preferable dosage range is fromabout 0.5 μg/kg to about 0.15 mg/kg of patient body weight. A suitabledose size in humans is about 0.1-1 ml, preferably about 0.1 ml.Accordingly, a dose for intramuscular injection, for example, wouldcomprise 0.1 ml containing immunogen in admixture with 0.5% aluminumhydroxide.

The vaccine can be administered to pregnant women or to women of childbearing age to stimulate maternal antibodies. The female can berevaccinated as needed. Infants can be vaccinated at 2 to 3 months ofage after depletion of maternal antibodies and revaccinated asnecessary, preferably at 6 to 9 months of age after maturation of theimmune system. Babies born to unvaccinated mothers can be vaccinated at2 to 3 months of age. The vaccine may also be useful in othersusceptible populations such as elderly or infirmed patients.

The vaccine may also be combined with other vaccines for other diseasesto produce multivalent vaccines. It may also be combined with othermedicaments such as antibiotics. ##STR11##

I claim:
 1. An expression system comprising a suitable host containing aDNA sequence capable of expressing a chimeric glycoprotein consistingessentially of the extracellular domain of parainfluenza virus type 3Fprotein linked to the extracellular domain of parainfluenza virus type 3HN protein.
 2. The expression system according to claim 1 wherein saidsuitable host is selected from group consisting of yeast cells,mammalian cells and insect cells.
 3. The expression system according toclaim 2 wherein said suitable host is selected from the group consistingof Chinese hamster ovary cells, murine C127 cells and S. Frugipersacells.
 4. The expression system according to claim 1 wherein saidsuitable host secretes said chimeric glycoprotein.
 5. The expressionsystem according to claim 1 wherein said DNA sequence is contained in aplasmid.
 6. The expression system according to claim 5 wherein saidplasmid is under the control of a cytomegalovirus promoter.
 7. Theexpression system according to claim 5 wherein the replication of saidplasmid while in a suitable eukaryote host is under the control ofbovine papilloma virus DNA sequences.
 8. The expression system accordingto claim 1 wherein said DNA sequence is contained in a recombinant virusof the baculovirus family.
 9. The expression system according to claim 8wherein the virus is Autographs californica nuclear polyhedrosis virus.